In order to successfully treat a disease caused by a bacterium the rapid and accurate detection and identification of the disease-causing bacterium is required. Bacterial detection and identification have traditionally been accomplished by pure culture isolation, followed by identification procedures that make use of knowledge of specimen source, growth requirements, visible (colony) growth features, microscopic morphology, staining reactions, and biochemical characteristics.
An important step in determining the identity of a bacterium is the Gram stain. This procedure involves treating a heat-fixed bacterial smear on a glass slide with the basic dye, crystal violet. All organisms take up the dye. The smear is then covered with Greta's iodine solution (3 percent iodine-potassium iodide in water or a weak buffer, pH 8.0, in order to neutralize acidity formed from iodine on standing). After a water rinse and decolofization with acetone, the preparation is washed thoroughly in water and counterstained with a red dye, usually safranin. The stained preparation is then rinsed with water, dried, and examined under oil using a light microscope.
Most bacteria can be differentiated into two groups by this stain. Gram-positive organisms stain blue, whereas about one-third of the cocci, one-half of the bacilli, and all spiral organisms stain red and are said to be gram-negative. This method, while effective, is very time consuming and involves many different procedures which present many opportunities for error. For blood samples the Gram stain and other culture-based methods of detection require incubation of the sample with culture medium at least overnight in order to obtain a pure culture.
The presence of bacteria or fungi in the blood, commonly referred to as septicemia, can have severe and life-threatening clinical consequences. Septicernie can result in septic shock, which includes the following symptoms--hypotension, lactic acidosis, hypoxemia, oligouria, confusion, disseminated intravascular coagulation, gastrointestinal bleeding, disturbances of metabolism, and subtle skin lesions. As little as one colony-forming unit (CFU) may be present in a 30 ml blood sample in a patient with septicemia. Since culture is currently the most sensitive and commonly used method of detecting bacteria or fungi in the blood, treatment of suspected septicemia is often begun empirically, without waiting for the results of culture. It is clear that a rapid diagnostic method for detecting bacteria in the blood with the same sensitivity as culture would be a significant improvement over currently used methods.